实时定量PCR检测胃癌组织miR-20a及初步应用

目的建立检测人胃癌组织中miR-20a的实时荧光定量PCR方法。方法建立miR-20a标准曲线,评价该法检测的线性范围及灵敏度;对标准质粒扩增产物进行熔解曲线分析和琼脂糖凝胶电泳检测以分析该法的特异性。取高、中、低3份不同浓度的标准质粒进行批内和日间变异系数(CV)的检测,分析该法的重复性。收集70例胃癌患者手术切除的癌组织和对应的癌旁组织,定量检测组织中miR-20a的表达水平。结果构建的实时荧光定量PCR法的灵敏度为101copies/μL,扩增的线性范围为101～109copies/μL;扩增产物的熔解曲线峰和电泳片段特异;3份不同浓度标准质粒定量检测的批内CV分别为6.33%、4.74%、5.89%,日间CV分别为9.45%、6.29%、7.48%。定量结果显示75.7%(53/70)胃癌组织中miR-20a的表达高于癌旁组织,差异有显著性(Z=-4.427,P<0.01)。结论建立的实时荧光定量PCR检测miR-20a的方法准确、快速、灵敏、特异,可用于胃癌组织中miR-20a的检测。

A real-time quantitative PCR for detection of miR-20a and its application

Objective To establish a real-time quantitative PCR for detecting miR-20a in gastric cancer tissues.Methods miR-20a standard plasmids were established by a series of dilution,and used to evaluate the dynamic range,sensitivity and specificity of this method.The reproducibility was determined by intra-and inter-assay of miR-20a in three different standard plasmids for five times.Seventy pairs of gastric cancer tissues and adjacent non-tumor tissues were collected and the levels of miR-20a in these tissues were detected by real-time quantitative PCR.Results The developed method in this study could precisely detect 10 copies/μL of miR-20a,and had a dynamic range from 101 to 109 copies/μL.The intra-assay CV for three different standard plasmids were 6.33%,4.74% and 5.89%,while the intra-assay CV were 9.45%,6.29% and 7.48%.The level of miR-20a expression in 75.7%(53/70) gastric cancer tissues was significantly higher than that in adjacent non-tumor tissues(Z=-4.427,P<0.01).Conclusion An accurate,rapid,sensitive and specific real-time PCR assay was successfully established to quantify miR-20a in gastric cancer tissues.miR-20a up-regulation in gastric cancer may be associated with generation and progression of gastric cancer.